Characterisation of a haemolysin produced by Vibrio splendidus, a pathogen of turbot larvae

• Main Author: Macpherson, Hazel Louise
• Format: Thesis
• Language: English
• Published: 2004
• Subjects: Turbot
• Online Access: http://theses.gla.ac.uk/71089/; http://theses.gla.ac.uk/71089/1/10390658.pdf; https://eleanor.lib.gla.ac.uk/record=b2224651

Although turbot is produced in substantial quantities in aquaculture in Europe the major limitation in expansion of the industry is the supply of juvenile fish for on- growing. High mortalities can occur during early life stages during the critical times of transition from yolk sac larvae to feeding on rotifers and Artemia. There is good evidence that microorganisms are the major cause of the problems associated with early-feeding, as improved survival is seen when antibiotics are used or when larvae are reared in bacteria-free conditions. Several studies have shown that the bacterial flora of the larval turbot gut originates from the food. However, high levels of certain bacteria in the gut flora are not harmful, rather it is particular bacterial types that appear to determine larval rearing success. The present study concentrated on determining the virulence mechanisms of Vibrio splendidus DMC-1 (biovar 1), which was isolated from a batch of turbot larvae suffering very high mortality at a turbot hatchery. This isolate was shown to be lethal in turbot larvae challenge assays with immunohistochemistry studies of challenged larvae showing signs of enteritis in the gut. A protein of c. 40 kDa with an N-terminal amino acid sequence showing homology with the OmpU outer membrane protein from V. vulnificus, but other potential virulence factors secreted by this organism were investigated. Vibrio splendidus DMC-1 grown in Marine Broth produced high titres of haemolysin and haemagglutinin against turbot blood. The haemagglutinin appeared specific for turbot cells and may function as an adhesin. The haemolysin had a broad spectrum of cell-damaging activity and was partially purified and shown to be a heat-labile pore-forming protein toxin, producing membrane channels of approximately 1.8 nm diameter. The toxin was secreted into the culture medium during logarithmic growth with production being subject to catabolite repression, with medium composition governing the level of haemolysin produced. Transposon mutagenesis was used to identify the haemolysin gene. Over 10,000 mutants were screened, resulting in isolation of 3 fully haemolysin-negative (hly-) mutants and several with reduced expression of haemolysin. Two different hly- mutants that were non-haemolytic, non-cytotoxic to turbot tissue culture cells and avirulent against turbot larvae were characterised. Over 8 kb of the V. splendidus genome was sequenced to identify the haemolysin gene and neighbouring genes. Overall, the DNA sequence had high homology with sections of the genomes of V. cholerae, V. parahaemolyticus and V. vulnificus, with the same genes and gene order as in these species. In addition, Vibrio splendidus contained a section of DNA inserted into this common Vibrio background that was unrelated to the above vibrios but that closely resembled the gene for aerolysin, a pore-forming cytotoxin from the Aeromonas genus that has not previously been recognised outside that genus. The predicted molecular weight of this toxin was 55 kD. The presence of this toxin could explain the enteritis found in the larval turbot gut as the haemolysin-negative transposon mutant lacked the ability to cause cell damage in vitro and in vivo. In the second haemolysin-negative mutant the transposon insertion site was in a gene adjacent to that for the cytotoxin. The translation product of this gene showed homology with a well-established group of regulators of bacterial virulence gene expression, known as ToxR. Analysis of the secondary structure of this regulator indicated that it possesses all the domains necessary for gene regulation but with novel features not previously recorded for other regulators of virulence genes. As transposon mutagenesis of the ToxR-like gene causes loss of haemolytic activity it suggests that this gene is functional and controls expression of the haemolysin, but it is not known whether other genes are also regulated by this system.